Role of nerve growth factor on cognitive impairment in patients with Alzheimer's disease carrying apolipoprotein E ε4

Abstract Aims To investigate the roles of neurotrophic factors on cognition in patients with Alzheimer's disease (AD) carrying Apolipoprotein E (APOE) ε4. Methods Totals of 173 patients with AD were divided into APOE ε4 carrier and non‐carrier groups, and their demographics, cognition, and neurotrophic factors in cerebrospinal fluid (CSF) were compared. Multiple linear regression analyses were performed to assess correlations among APOE ε4, neurotrophic factors and cognition. Mediation analyses were conducted to assess the sequential associations among APOE ε4, nerve growth factor (NGF), and cognition. Results Global cognition and multiple domains were impaired in the APOE ε4 carrier group (all p < 0.05). NGF level in the APOE ε4 carrier group was lower than that in the non‐carrier group (p = 0.016). NGF level showed significant correlations with both global and multiple domains cognitions. Specifically, NGF mediated the association between APOE ε4 and Animal Fluency Test score (β, −0.45; 95% CI [−0.96, −0.07]; p < 0.001) and Trail Making Test‐A (time) (β, 0.15; 95% CI [0.01, 0.33]; p < 0.001). Conclusion APOE ε4 is associated with cognitive impairment, and those carrying APOE ε4 have decreased NGF level in CSF. Declined NGF level is correlated with compromised cognition. NGF mediates APOE ε4‐associated cognitive impairment.


| INTRODUC TI ON
Alzheimer's disease (AD) is the most common cognitive disorder among the elderly.Typical patients with AD are initially characterized by episodic memory decline, followed by overall cognitive impairment, neuropsychiatric symptoms, and impaired activities of daily living. 1 Apolipoprotein E (APOE), encoding the indispensable lipid transporter APOE protein in the brain, includes three alleles of ε2, ε3, and ε4, of which, APOE ε4 is the strongest risk gene for developing sporadic AD. 2 APOE ε4 carriers exhibit an earlier age of onset, a faster rate of cognitive decline, and a poorer cognitive outcome. 2,3Previous studies have linked APOE ε4 to synaptic dysfunction and aggravated cognitive impairment by promoting depositions of neuropathological proteins of AD 4,5 and eliciting neuroinflammation. 6However, the role of APOE ε4 on the neurotrophic state in the brain remains unclear.
Neurotrophic factors constitute a family of proteins, including brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor (NGF).These factors played crucial roles in the survival, growth, and differentiation of neurons during development. 7Decreased levels of BDNF and its precursor were observed in several brain regions and peripheral blood of patients with AD. 8 GDNF level was elevated in the cerebrospinal fluid (CSF) of early-stage patients with AD but decreased in the middle temporal gyrus and serum of patients with AD. 9 In addition, proNGF levels in the cerebral cortex and CSF of patients with AD were increased, while NGF mRNA was unchanged in the cerebral cortex of patients with AD. [10][11][12] It came to the limelight that abnormal secretions of these neurotrophic factors were correlated with the progression of neuropathology and cognitive impairment of patients with AD. 13,14 However, the relationships between neurotrophic factors in CSF, APOE ε4, and functions of different cognitive domains remain unknown.
In this study, we hypothesized that both APOE ε4 and neuro-

| Ethics statement
The study was approved by the Ethical Review Board of Beijing Tiantan Hospital.Written informed consent was obtained from patients and their caregivers.All the procedures were conducted in accordance with the guidelines and regulations of ethical principles for medical research involving human subjects of the Declaration of Helsinki.

| Participants
Patients diagnosed with AD according to the National Institute of Aging and Alzheimer's Association (NIA-AA) criteria 15,16

| Collection of demographic variables
Demographic variables, including gender, age, age of onset, disease duration, education level, drinking, smoking, and body mass index (BMI), were collected.In addition, a history of hypertension, hyperlipidemia, myocardial infarction, atrial fibrillation, diabetes mellitus, et al., was also collected.

| Assessment of cognitive function
The overall cognitive function of patients with AD was assessed by the Mini-Mental State Examination (MMSE) 17 and the Montreal Cognitive Assessment (MoCA). 18In the individual cognitive domains, verbal memory was evaluated by the Auditory Verbal Learning Test (AVLT), and visual delayed memory was evaluated by the Rey-Osterreithm Complex Figure Test (RCFT)-delayed recall. 19,20The language was evaluated by the Animal Fluency Test (AFT), the Verbal Fluency Test (VFT), and the Boston Naming Test (BNT). 21,22Attention was evaluated by the Symbol Digit Modalities Test (SDMT), 23 the Trail Making Test (TMT)-A, 24 as well as the Stroop Color-Word Test (SCWT)-A and the SCWT-B. 25suospatial ability was evaluated by the RFT.Executive function was evaluated by the SCWT-C and the TMT-B.A detailed description of these rating scales was provided in the Supplementary material.

| Detections of APOE genotypes
The venous blood samples of patients with AD were collected from the median elbow under fasting conditions in the morning following admission and then sent to the clinical laboratory of our hospital.Genotyping for APOE single nucleotide variants (rs429358 C/T and rs7412 C/T), which define APOE 2, 3, and 4, was performed by real-time fluorescence quantitative polymerase chain reaction using nucleic acid detection reagents (Youzhiyou company, Wuhan, China). 26

| Collections of CSF samples
Patients were requested to withdraw anti-cognitive impairment drugs for at least 12-14 hours before lumbar puncture if their conditions allowed.CSF samples were collected under fasting condition through lumbar puncture, followed by being immediately centrifuged at 4°C with 1500 g for 10 min.Each CSF sample was then allocated into separate Nunc cryotubes (Beijing JingkeHongda Biotechnology Co., Ltd, Beijing, China) and frozen for 0.5 mL per tube at −80°C until the assay.

| Demographic variables in patients with AD
A total of 173 patients with AD were enrolled in this study, of whom 55 cases (31.79%) carried APOE ε4, 34 cases (61.83%) were female, the mean age was 65.49 ± 9.00 years old, and the median disease duration was 24.00 (12.00, 48.00) months in APOE ε4 carrier group.Demographic variables, including gender, age, age of onset, disease duration, education level, smoking, drinking, BMI, etc., were not significantly different between the two groups (Table 1).

| Association of APOE ε 4 with cognitive function
A comparison of cognitive function between APOE ε4 carrier and APOE ε4 non-carrier groups was presented (Table 2)

| Association of APOE ε 4 with the levels of neurotrophic factors in CSF
The levels of neurotrophic factors in CSF were compared between APOE ε4 carrier and APOE ε4 non-carrier groups.NGF level in APOE ε4 carrier group was significantly lower than that in APOE ε4 noncarrier group (p = 0.016) (Figure 1).3).

| Association between NGF level in CSF and cognitive function
Multiple linear regression analyses were conducted to explore the association between NGF level in CSF and cognitive function in patients with AD after adjusting for age, gender, age of onset, disease duration, education level, and BMI (Figure 2 S2).

| DISCUSS ION
9][30] Nonetheless, the precise relationships between APOE ε4 and functions of cognitive domains remained elusive.9][30] In addition to memory, our novel observation indicated a significant association between APOE ε4 and impaired cognitive domains related to language and attention/executive function.

| Relationship between APOE ε 4 and cognitive impairment in patients with AD
These findings might be due to the faster rate of cognitive decline and poorer cognitive function that APOE ε4 carriers had. 2,3

| Relationship between APOE ε4 and the levels of neurotrophic factors in CSF in patients with AD
Within the scope of various neurotrophic factors measured in this study, NGF level in CSF from patients with AD with APOE ε4 was significantly declined compared to those without APOE ε4.APOE, a pivotal lipid transport protein in the brain, is secreted primarily by astrocytes and to a lesser extent by microglia.APOE binds with two major metabolic receptors, the low-density lipoprotein receptor and lipoprotein receptor-related protein, facilitating lipid transport between cells, redistributing intracellular lipids, and maintaining lipid balance in the brain. 31Compared to other APOE isoforms, APOE4 encoded by APOE ε4 exerted the weakest effect on lipid transport. 32olesterol homeostasis is essential for the brain, with insufficient accumulation leading to abnormal cellular nutrition and energy metabolism, and excessive accumulation resulting in synaptic plasticity damage and neuronal apoptosis. 33However, limited studies directly address the relationship between APOE ε4 or APOE4 and NGF.
Based on the results of this investigation, we speculate that the impaired cellular nutrient and energy metabolism, stemming from the weakened lipid transport capacity of APOE4, may contribute to the decrease of NGF level in CSF of patients with AD.

| Relationship between NGF level in CSF and cognitive impairment in patients with AD
In this study, we found a close relationship between NGF level in CSF

| Limitations
This study had limitations.First, while measuring neurotrophic factors in CSF is one of the most objective ways to capture their changes in the brain, it is tough to obtain CSF from elderly patients with AD, particularly from people with normal cognition.We will focus on collecting more CSF samples from a larger cohort of patients with AD and cogni-

| CON CLUS ION
The results from this study indicate a significant association be- trophic factors might be related to cognitive impairment, APOE ε4 might be associated with decreased neurotrophic factors in CSF, and the declined neurotrophic factors might play a pivotal role in mediating APOE ε4-associated cognitive impairment.Based on this hypothesis, patients with AD were divided into APOE ε4 carrier and APOE ε4 non-carrier groups.Demographic variables were collected, overall cognitive function and individual cognitive domains were assessed by a series of rating scales, and the levels of neurotrophic factors, including BDNF, GDNF, and NGF in CSF were measured by enzyme-linked immunosorbent assay (ELISA) with aims to figure out the roles of neurotrophic factors on cognitive impairment in patients with AD carrying APOE ε4.
were consecutively enrolled from the Center for Cognitive Neurology, Department of Neurology, Beijing Tiantan Hospital, Capital Medical University.The exclusion criteria included: (1) Patients with a history of diseases that might affect cognitive function besides AD, such as cerebrovascular diseases, Lewy body diseases, frontotemporal neurodegeneration, corticobasal degeneration, multiple sclerosis, epilepsy, substance abuse, etc. (2) Patients with severe systematic diseases, active infections, chronic wasting disease, autoimmune diseases, hematological system diseases, and malignant tumor.(3) Patients suffered from traumatic brain injury and major surgery recently.(4) Patients were undergoing steroid treatment.(5) Patients were unable to cooperate with all the examinations for various reasons.

| 3 of 9 HE
et al.
Statistical analyses were performed by SPSS Statistics 25.0 (IBM Corporation, New York, USA).Statistical significance was defined as a two-sided p < 0.05.The data were tested for normal distribution using the Kolmogorov-Smirnov test.Demographic variables, cognitive function, and neurotrophic factors in CSF were compared between APOE ε4 carrier and non-carrier groups.Continuous variables conforming to normal distribution were presented as means ± standard deviations (SD) and compared by two-tailed t-test, while non-normal distributed variables were presented as median (quartile) and compared by a non-parametric test, and categorical variables were presented as number (percentage) and compared by Chi-Squared test.Multiple linear regression analyses were performed to assess the correlations among APOE ε4, neurotrophic factors in CSF, and cognitive function of patients with AD.Mediation analysis was conducted to evaluate the sequential associations among APOE ε4, NGF, and cognitive function.
and the cognitive function of patients with AD.It is well known that the main source of acetylcholine in hippocampus and cerebral cortex is the forebrain cholinergic neurons, which dysfunction or degeneration is the prominent cause of decreased acetylcholine level and impaired cognitive function in patients with AD. 1 NGF, a member of the neurotrophin family, supplies the forebrain cholinergic neurons to maintain their cholinergic phenotype.Degeneration of the cholinergic system is associated with dysregulation of the NGF metabolic pathway, which controls the maturation and degradation of NGF.34 Previous studies have indicated that the disturbed regulation of NGF pathway, including the impaired maturation and increased degradation of NGF, existed in AD, even in the preclinical stage of the disease.35,36NGF deprivation was associated with poorer cognitive function, and treatment with NGF ameliorated neuropathology F I G U R E 1 The levels of neurotrophic factors in CSF from APOE ε4− and APOE ε4+ groups.Comparison of the levels of BDNF (A), GDNF (B), and NGF (C) in CSF between APOE ε4− and APOE ε4+ groups.APOE ε4−, APOE ε4 non-carriers; APOE ε4+, APOE ε4 carriers.APOE, apolipoprotein E; BDNF, brain-derived neurotrophic factor; CSF, cerebrospinal fluid; GDNF, glial cell-derived neurotrophic factor; NGF, nerve growth factor.*p < 0.05.

TA B L E 3 4 . 4 |
Association between APOE ε4 and neurotrophic factors in CSF in patients with AD. and thereafter inhibited memory decline in AD animal models.11,37However, the relationships between NGF and functions of different cognitive domains remain unclear.In this study, we observed, for the first time, a strong association between NGF level in CSF and impaired global cognition and functions of multiple cognitive domains, including memory, language, attention, and executive function.In addition, the declined NGF level was particularly and fairly associated with compromised memory and language in patients with AD carrying APOE ε4.At the early stage of AD, cognitive domains, such as memory and language, are specifically vulnerable to the decrease of NGF in the individuals carrying APOE ε4, as indicated by the results from this study.The detailed mechanisms underlying their association warrant further investigation in the future.Role of NGF on APOE ε 4-associated cognitive impairment in patients with AD Previous studies highlighted that APOE ε4 exacerbated cognitive impairment by promoting the depositions of neuropathological proteins in AD and facilitating neuroinflammation by activating microglia and astrocytes.[4][5][6]However, the roles of neurotrophic factors on APOE ε4 and the associated cognitive impairment remain unclear.In the current study, we for the first time found that the declined NGF level in CSF played an important role in mediating APOE ε4-associated impairments of overall cognition and function of cognitive domains, particularly language and attention/executive function.Given that APOE4 has poorer lipid transport capacity than other APOE isoforms, leading to lipid instability in cells, we speculate that APOE4 may be responsible for the declined NGF level in the brain, and NGF deficiency may further accelerate degeneration of the cholinergic system, eventually propagating cognitive impairment in patients with AD.The potential mechanisms illustrating the mediating role of NGF on APOE ε4-associated impairments of language and executive function remain to be elucidated.

tween
APOE ε4 and compromised cognitive function in patients with AD.Patients with AD carrying APOE ε4 have prominently decreased NGF level in CSF.Decreased NGF level is markedly associated with drastically impaired overall cognition and individual cognitive domains, including memory, language, and attention/executive function.More importantly, NGF plays a pivotal role in mediating remarkably compromised cognitive function associated with APOE ε4.These novel findings contribute to a better understanding of the mediating role of NGF deficiency on cognitive impairment of patients with AD carrying APOE ε4, thus, replenishing NGF may be an effective therapy for slowing down cognitive impairment in patients with AD carrying APOE ε4.AUTH O R CO NTR I B UTI O N S Mingyue He and Zhan Liu contributed to the conception, design, and data statistics of the study and paper writing; Tenghong Lian, Peng Guo, Wenjing Zhang, Weijiao Zhang, Jinghui Li, Huiying Guan, Weijia Zhang, Dongmei Luo, Jing Qi, and Hao Yue contributed to the acquisition and collation of data; Yue Huang, Yanan Zhang, Gaifen Liu, and Xiaomin Wang contributed to directing paper writing and data statistics; Wei Zhang contributed to the conception, design and implementation of the study and the supervision of paper writing.
Note: Age, gender, age of onset, disease duration, education level, and BMI were adjusted.
Association between NGF level in CSF and cognitive function in patients with AD.Multiple linear regression analyses were performed between NGF level and MMSE (A), RCFTdelayed (B), AFT (C), VFT-alternating fluency (D), TMT-A (time) (E), and SCWT-B (F) in patients with AD after adjusting for age, gender, age of onset, disease duration, education level, and BMI.AD, Alzheimer's disease; AFT, Animal Fluency Test; CSF, cerebrospinal fluid; MMSE, Mini-Mental State Examination; NGF, nerve growth factor; RCFT, Rey-Osterrieth Complex Figure Test; SCWT, Stroop Color and Word Test; TMT, Trail Making Test; VFT, Verbal Fluency Test.
normal controls.Second, unlike a randomized controlled design, this cross-sectional study may introduce relative selection bias.F I G U R E 2